This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Apoptosis is a cell death program critical for orchestrating development and maintaining tissue homeostasis of multi-cellular organisms. Aberrant blockade of apoptosis is associated the development and progression of cancer. On the other hand, most cancer therapeutics function by inducing apoptosis in cancer cells. The mitochondria-dependent pathway is primarily responsible for apoptosis, and is largely regulated by the Bcl-2 family proteins at the step of mitochondrial outer membrane permeabilization (MOMP). The mechanism of Bax/Bak-dependent MOMP during apoptosis remains poorly understood. Our preliminary studies found that active mutants of Bax or Bak are potent inducers of bacterial cell lysis and cell death. More importantly, they function in a fashion similar to that of bacteriophage holin. Our hypothesis is that active Bax functions as a holin to cause bacterial membrane lesions. In Aim 1, we propose to examine the structural elements of Bax that are mediating the bacterial lysis activity. In Aim 2, we propose to examine the requirement of homo-oligomerization for the bacterial lysis activity of active Bax. In Aim 3, we propose to examine the bacterial inner membrane localization of active Bax, and identify the bacterial membrane targeting sequence of Bax. In Aim 4, we propose to test the functional and genetic replacement of holin in lamda bacteriophage. These studies are expected to mechanistically linked MOMP to holin-mediated hole formation in the bacterial membrane. If successful, this work will provide novel insights into the mechanism of MOMP, and establish bacteria as a powerful model system for the study of apoptosis.